Within this application several references are referenced by arabic numerals within parentheses. Full citations for these publications may be found at the end of the specification immediately preceding the claims. The disclosures of all of these references in their entireties are hereby expressly incorporated by reference into the present application.
Many of the basic electronic properties of metals can be described by modeling them as condensed matter plasmas. Their electrons behave as a high density gas in a lattice of fixed positive charges. In this analogy, longitudinal density fluctuations, (i.e., plasma oscillations), can propagate through the volume of the metal. These oscillations, "volume plasmons," are quantized with an energy hw.sub.p where w.sub.p is the plasma frequency of the metal: ##EQU1## where n is the volume electron density, e is the electron charge, and m.sub.e is the electron mass. The permittivity of a metal (.epsilon..sub.m) is a function of its plasma frequency and electron damping. It generally has the complex form .epsilon..sub.m (.omega..sub.p)=.epsilon..sub.re (.omega..sub.p)+i.epsilon..sub.im, where the imaginary component is related to the damping in the metal.
Electrons at the surface of a plasma behave somewhat differently from the "volume" electrons in the center of a plasma, due to the discontinuity at the interface. They still, however, exhibit coherent density fluctuations, which are known as surface plasmon oscillations. Surface plasmons can only exist at the interface between a metal film (.epsilon..sub.m), 10, and a dielectric film (.epsilon..sub.d), 20, as shown in FIG. 1(a). The magnitude of the electric field of these charge fluctuations, shown in FIG. 1(b), is a maximum at the interface (z=0) and decays exponentially away from the surface. The wave propagates along the interface in the x-direction with a wave vector whose magnitude is k.sub.sp =2.pi./.lambda..sub.sp, where .lambda..sub.sp is the wavelength of the surface plasmon oscillation and h.omega..sub.spp is the quantized energy of the excitation. FIG. 1(a) illustrates a metal/dielectric interface that supports surface plasmon waves and the exponential decay of the electric field distribution: EQU E=E.sub.0.sup.= exp[i(k.sub.sp x.+-.k.sub.z z-.omega..sub.spp t)](1b)
here + indicates z.gtoreq.0, - indicates z&lt;0, and k.sub.z is imaginary.
Surface plasmons are only one variety of many possible elementary quantized excitations of solid state matter, such as phonons, polaritons, excitons, and magnons. A polariton is defined as the coupled state between a photon and an excitation quantum. As the name polariton implies, these "quasiparticles" can only be excited using polarized light. A surface plasmon polariton (SPP) is the coupling of oscillating surface charge density with an electromagnetic field. Since surface plasmon waves propagate in a TM (transverse magnetic) mode, TM-polarized light will excite SPPs. Reference (1) discusses many of these properties in detail. The dispersion relation for the surface plasmon mode is a function of the permittivities of the metal (.epsilon..sub.m) and dielectric materials (.epsilon..sub.d) at the interface. The surface plasmon wave vector (k.sub.sp) is defined by the dispersion relation as: ##EQU2## where c is the speed of light in vacuum. Incident light cannot couple directly to surface plasmons on smooth surfaces since the real part of the radical in Eq. (2) is always greater than unity. Several techniques exist to excite to surface plasmons, such as electron beam bombardment, grating coupling, and total internal reflection (hereinafter, TIR) prism coupling.
If light is incident on the internal surface of a prism, 30, at an angle greater than the critical angle for total internal reflection, as shown in FIGS. 2(a) and 2(b), an evanescent wave is produced below the reflecting surface, 32, of the prism, 30. The evanescent wave decays exponentially in the direction normal to the prism surface, 32, (into the metal film, 10, / dielectric film, 20, bilayer) and has a dispersion relation given by: ##EQU3## where n.sub.p is the refractive index of the prism, .omega. is the frequency of the incident light, .theta. is the incidence angle of light on the surface of the prism, and c is the speed of light in vacuum. For this technology, the prism is replaced by a novel "substrate" mode waveguide. The evanescent field dispersion relation at the reflecting interface is the same as Eq. (3), with n.sub.p replaced by n.sub.wvg, the index of refraction of the waveguide.
FIG. 2(a) illustrates surface plasmon resonance through prism coupling. FIG. 2(b) illustrates reflectance near plasmon excitation. The resonant condition where the curves intersect, i.e. k.sub.x =k.sub.sp and hw=h.omega..sub.spp, is analytically described by combining the surface plasmon dispersion relation, Eq. (2) and Eq. (3) to give: ##EQU4## which shows that the index of refraction of the prism and the permittivities of the thin film layers determine the incidence angle for which surface plasmon polaritons can be excited.
FIG. 2(b) shows schematically the reflected intensity of TM-polarized light from the prism as a function of incidence angle. The dip in reflectance is due to the absorption of energy from the evanescent wave by surface plasmons. Coupling of the light from the prism to the SPPs attenuates the amount of light totally internally reflected from the prism and dissipates it in the metal film as thermal energy. The phenomenon is strongest for incidence angle .theta..sub.sp, where maximum coupling occurs and the reflectivity is nearly zero. At a typical interface, surface plasmons propagate approximately 10 .mu.m before their energy is dissipated as heat. This short propagation length enables highly localized sensing. The nature of the resonance shown in FIG. 2(b) is revealed by analytically solving for reflectivity. By so doing, the Lorentzian nature of the plasmon resonance is revealed: ##EQU5## where .GAMMA..sub.i =Im(k.sub.sp), .GAMMA..sub.rad =Im(.DELTA.k.sub.x), .DELTA.k.sub.x =.function.(.epsilon..sub.m, .epsilon..sub.d, t.sub.i), and t.sub.i is the thickness of the metal thin film layer in the structure. See reference (10). Eq. (5) reveals that the thickness of the metal film determines the depth of the resonance by the minimum condition .GAMMA..sub.i =.GAMMA..sub.rad. Only for one particular film thickness will the resonance completely attenuate the incident TM polarized light at the excitation angle .theta..sub.sp.
The previous discussion elucidates two fundamental mechanisms by which surface plasmons can be used for sensing: the first is to fix the angle of incidence of TM-polarized light at .theta..sub.sp so that virtually no light will be transmitted. If the permittivity of one of the layers (e.g. the dielectric layer) is varied, the angular position of the plasmon resonance minimum shifts and the amount of transmitted light increases; second, if the thickness of one of the layers is varied, the efficiency of the coupling is modulated and the transmitted light again increases. By incorporating a dielectric transducing layer whose permittivity and/or thickness varies in response to analytes of interest, the surface plasmon multilayer can become a sensor.
Immunoassays and nucleic acid (DNA/RNA) probes are analytical techniques that use antibody-related reagents and nucleic acid hybridization, respectively, for the selective determination of sample components. For immunoassay, by monitoring the amount of antibody-to-antigen binding that occurs for a fixed amount of antigen or antibody, the concentrations of target analytes are determined. With nucleic acid probes, monitoring the selective binding (hybridization) of target DNA or RNA with genetic probes allows determination of the amount of target analyte. These techniques have the advantage of high selectivity, low detection limits, and compatibility with use in complex samples such as urine and blood, or soil samples in the case of bioagent detection. Immunoassay and detection technologies based on radiometric, enzymatic, surface acoustic wave, and optical means have been investigated for many years. See references (2, 3). Of these, optical techniques promise greater sensitivity, lower detection limits, and higher flexibility. Immunoassay and nucleic acid probe detection based on such optical techniques as attenuated total reflection (hereinafter, ATR), surface plasmon resonance (hereinafter, SPR), fluorescence, and bio/chemiluminescence have been studied and commercial immunosensors based on some of these techniques are beginning to appear. See references (4, 5).
The optical techniques are differentiated by whether they use direct or indirect sensing methods. Indirect biosensing uses secondary labels that are attached to the analyte of interest in a separate step, before binding, to differentiate between specific and non-specific binding events. Optical labels include organic fluorophores and bio/chemiluminescent molecules. These labels emit light whose intensity is quantified as a measure of the amount of analyte present. If no tagged analytes are present then, theoretically, no signal is measured. In the case of fluorescence immunoassay, an external source is required to cause the labels to fluorescence. This has several disadvantages since the excitation light must be kept very stable and strongly post filtered in order to achieve high sensitivity. For low analyte levels (corresponding to low fluorescent light levels) the background of the excitation light that makes it through the filter (none are 100% efficient) can exceed the low level fluorescent signals, thus limiting the lower detection limit of a fluorescence biosensor. Direct biosensing involves measuring the direct antigen/antibody binding, or DNA/RNA hybridization, without the use of any secondary labels. Because of this, direct techniques promise more rapid screening times due to the reduced number of assay steps, and commensurate reduction in operator skill, required to perform reliable tests. Though the direct techniques (ATR and SPR) promise more rapid screening by reducing immunoassay steps, they can suffer from interference by non-specific binding in the presence of proteins or other interferents.
Immunoassay biosensors monitor the specific binding that occurs between antibodies and antigens. Antibodies are simply proteins produced by the lymphocytes of higher animals in response to the presence of foreign molecules (antigens). These proteins have been synthesized for a wide variety of antigens by introducing the antigen into a host and isolating the corresponding antibodies produced by the lymphocytes in the host's immune system. See reference (6). More recently, the production of large amounts of specific antibodies by genetically engineered microorganisms has been reported. Sensitive immunological detection of both biological and chemical materials has been demonstrated. See reference (7).
Because each immunological binding event involves two different molecules, immunoassay can be used to perform two primary tasks: detection of antibodies using antigens, and detection of antigens using antibodies. The detection of antibodies is of primary interest in biomedical diagnostics, where the presence of antibodies associated with a particular disease, bacteria, or virus indicates the presence of the infection. For environmental application of immunoassay, detection of antigens is required since these are the toxic agents that disable personnel. As a result, environmental immunoassay biosensors require the development of antibodies to the biomaterials of interest before detection can be performed. This is in contrast to most biomedical applications, where a known antigen is often used to detect antibodies that are produced in the host's body in response to that same antigen, indicating the presence of the disease.
Nucleic acid probe hybridization (binding) provides a convenient way of detecting and measuring specifically defined nucleotide sequences in target analytes. By immobilizing a particular gene probe sequence, homologous gene sequences in target analytes bind specifically to the immobilized probe allowing direct detection of the target analyte. Gene probes can be made of either DNA or RNA and typically contain anywhere from 25 bases (nucleotides) to 10 kilobases. The target analyte can be upwards of a million bases in size. Since genetic information is highly specific, nucleic probes promise high sensitivity and specificity.
Heretofore surface plasmon resonance sensors were known. A conventional surface plasmon resonance sensor is typically based on a free space configured prism. For example, U.S. Pat. No. 4,844,613, the entire contents of which are hereby expressly incorporated herein, discloses an optical surface plasmon sensor device that uses a free space configured prism. U.S. Pat. No. 4,997,278, the entire contents of which are hereby expressly incorporated herein, discloses biological sensors based on surface plasmon resonance using a free space configured hemispheric prism. U.S. Pat. No. 5,064,619, the entire contents of which are hereby expressly incorporated herein, discloses biological sensors based on surface plasmon resonance using free space configured movable reflectors and a separate sensing surface chip that is index matched to a free space configured prism. Similarly, U.S. Pat. No. 5,055,265, the entire contents of which are hereby expressly incorporated herein, also discloses biological sensors based on surface plasmon resonance using free space configured movable reflectors. World Intellectual Property Organization International Publication Number WO 90/05305, the entire contents of which are hereby expressly incorporated herein, discloses a biosensor system based on surface plasmon resonance using a separate sensing surface chip that is index matched to a free space configured prism.
Surface plasmon biosensors are basically sensitive refractometers that monitor changes in the optical state of immunochemical or nucleic acid probe layers. This is accomplished by depositing a biosensing layer on top of a thin metal film evaporated onto the base of a TIR prism as shown in FIG. 3(a), which depicts an SPR immunoassay biosensor configuration. The SPR occurs at a specific angle of incidence that depends on the optical properties of the metal film, 10, and biosensing material, 40, coated on the metal film, 10. When TM-polarized light in the prism, 30, is incident at the proper angle to excite surface plasmons, the TM-polarized light is attenuated drastically. In the presence of a target bioagent, 50, the thickness and refractive index of the effective "dielectric" layer changes, thereby altering the angular position of the SPR as shown in FIG. 3(b). By measuring the shift in the plasmon resonance angle as a function of time (.DELTA..theta./.DELTA.t), the concentration of bioagent can be quantified.
A previously recognized problem concerning surface plasmon sensors has been that in order to sense the attenuated region, the angle of reflectance must be located accurately so as to be detected by photosensors. A previously recognized solution was to rotate either the light source or the prism so that the attenuated region would be incident upon a detector array. A drawback of this previously recognized solution is that complex optical and mechanical systems were required. Moreover, during the rotation, more binding of the analyte will occur, thereby altering the position of the attenuated region. To date, the only commercial SPR based biosensing system is an extremely bulky device that requires replacing two components each time a measurement is made and is totally unsuitable for use as a compact, handheld sensor system.